Ann. For. Sci. 59 (2002) 197–204 197 © INRA, EDP Sciences, 2002 DOI: 10.1051/forest:2002006 K.Dominance El Karkouri of R. et rubescens al. in a P. pinea plantation Original article Dominance of the mycorrhizal fungus Rhizopogon rubescens in a plantation of Pinus pinea seedlings inoculated with Suillus collinitus Khalid El Karkouria, Francis Martinb and Daniel Mousaina* a Laboratoire de Recherches sur les Symbiotes des Racines**, Institut National de la Recherche Agronomique, AgroM-INRA, 2 Place Viala, 34060 Montpellier Cedex 1, France b Unité Mixte de Recherche INRA-UHP Interactions Arbres / Micro-organismes, Centre INRA de Nancy, 54280 Champenoux, France (Received 15 January 2001; accepted 28 June 2001) Abstract – We examined the below-ground mycorrhizal diversity of P. pinea seedlings inoculated with S. collinitus six years after out- planting in a disturbed site, located at La Petite-Camargue (Gard, France). This was performed using the polymerase chain reaction (PCR), the restriction fragment length polymorphism (RFLP) and the sequencing of the nuclear ribosomal internal transcribed spacer (ITS). Seven and one plants corresponding, respectively, to inoculated and non-inoculated (control) treatments were chosen randomly. Examinations were carried out directly from ectomycorrhizae. A total number of 233 root tips was examined. Five ITS RFLP taxa were detected. The ITS taxon I corresponded to the ectomycorrhizal species Rhizopogon rubescens. This fungus was abundant (55%) in the P. pinea ectomycorrhizal community. The other ITS taxa were rare and remained unidentified. The current P. pinea plantation showed a restricted diversity of the ectomycorrhizal community which is typical of ectomycorrhizal communities in young plantations established in disturbed stands. Pinus pinea L. / plantation / ectomycorrhizal diversity / PCR-RFLP / rDNA (ITS) Résumé – Dominance de l’espèce mycorhizienne R. rubescens dans une plantation à Pinus pinea. La diversité ectomycorhizienne de plants de P. pinea, inoculés par S. collinitus, a été examinée six années après transplantation dans un site perturbé de La Petite-Ca- margue (Gard, France). L’identification de cette diversité fongique a été effectuée à partir de l’espaceur interne transcrit (ITS) de l’acide désoxyribonucléique (ADN) nucléaire et ribosomal. Deux marqueurs moléculaires dérivés de la réaction de polymérase en chaîne (PCR) ont été utilisés : le polymorphisme de fragments de restriction (RFLP) et le séquençage des produits d’amplification de l’ADN. Sept et un plants correspondant, respectivement, aux plants inoculés et non inoculés (témoin) ont été choisis au hasard. Le typage moléculaire a été réalisé directement à partir des ectomycorhizes. Deux cent trente trois racines courtes mycorhizées ou non ont été examinées. Cinq types d’ITS RFLP ont été détectés. Le type I correspond à l’espèce ectomycorhizienne Rhizopogon rubescens. Cette dernière, est domi- nante (55 %) dans la communauté ectomycorhizienne de cette plantation. Les autres types d’ITS RFLP ont été rares et restent non identi- fiés. La plantation de P. pinea a montré une faible diversité ectomycorhizienne qui est typique des communautés ectomycorhiziennes des jeunes plantations établies dans des sites perturbés. Pinus pinea L. / plantation / diversité ectomycorhizienne / PCR-RFLP / ADNr (ITS) * Correspondence and reprints Tel. 04 99 61 24 53; Fax. 04 67 54 57 08; e-mail: [email protected] ** Present address: Unité Mixte de Recherche INRA-ENSAM Sol et Environnement, Équipe Rhizosphère et Symbioses. 198 K. El Karkouri et al. 1. INTRODUCTION with S. collinitus (J 3.15.2) and non-inoculated (= con- trol) seedlings. This fungus was collected under a 20- year-old P. pinea plantation in a calcareous arenosol site In natural ecosystems, roots of forest trees live in as- at “La Grande-Motte” (south of France) in 1985. It was sociation with below-ground soil-born fungal communi- also distinguished from other Suillus spp. [16] and from ties and populations forming symbiotic structures called other S. collinitus strains [6] using ITS sequence and ectomycorrhizae (ECM) [11]. The ectomycorrhizal my- isozyme analysis, respectively. Each treatment was sub- celium of these symbiotic organs grows and develops or jected to a fertilization with a liquid fertilizer (formula 8- not fruit bodies above the ground. The ectomycorrhizal 6.5-13, Dynaflor, Sète, France) diluted at 0.5% (D), a symbiosis is known to improve the mineral nutrition, the fertilization with the same solution at 0.1% (D/5) and no growth and the adaptation of forest trees. Ectomycorrhizal fertilization (NF) in the nursery. The inoculated and the fungi have thus been exploited in many reforestation control P. pinea seedlings were introduced in three ran- programmes (for a review see [17, 18]). However, domized complete plots (I, II and III) in the plantation in knowledge of the structure and the composition of the January 1990. Only 55 plants of plot III (36 m × 24 m) in- ectomycorrhizal fungal communities (EFCs) of the oculated with S. collinitus (J 3.15.2) and the correspond- young plantations is still limited [10, 22]. In addition, ing 14 control plants, all fertilized with the D/5 solution, comparing to the number of recent reports performed on were considered for further sampling and DNA typing. the analysis of the EFCs from mature forests [e.g. 4, 5, 8, The inoculated and the control treatments were distrib- 13, 14, 23, 24], information on the EFCs in the young uted in three and two lines of plants, respectively. The plantations and in the young stands [3, 24] and their rela- two treatments, the lines and the plants were 9, 3 and tionships with those of the mature forests are rare. 1.5 m apart, respectively. In the Mediterranean region, the deterioration of for- ests and its consequences on soil conservation and the stress accompanying the outplanting of young forest 2.2. Sampling plants, roots and ECM trees are often related to the drastic edaphic and climatic conditions. Indeed, for example in the region of “La Pe- tite-Camargue” (Gard, France), the Brasinvert’s domain Soil was carefully removed starting near the base of of approximately 200 hectares of calcareous arenosol the plant stem until the roots appear. Digging was then was subjected to ecological disturbances (violent storms carefully continued in a centrifugal direction to the end and drought stress) before 1983, leading to the death of of the long roots of 0.97– 2.3 m of length. No abundant 90% of mature Pinus pinea trees between 1983 and 1986 roots and young mycorrhizal morphotypes were ob- [2]. After that disturbance, an experimental plantation served during root and ECM surveys. Two types of ECM was established in the domain in 1990 with nursery-non- were found: young ECM with well developed mantle inoculated and– inoculated with S. collinitus Pinus pinea and old ECM associated to dried black roots and having seedlings [2]. either no mantle or a naturally damaged mantle. A total of eight plants corresponding to 12.7% and 7% of respec- The present investigation examined the diversity of tively inoculated and control treatments were examined below-ground P. pinea EFC six years after outplanting in randomly (table I). The inoculated treatment was exam- this disturbed site. This was performed using PCR-RFLP ined in both Spring and Autumn 1996 (i.e. six years after and sequencing of the rDNA ITS directly from the outplanting), while control treatment was examined in ectomycorrhizal root tips. Spring of the same year. Nearly all the roots (4 to 9 per plant) and all the young ECM observed were sampled, while the old ECM were chosen randomly. Both roots 2. MATERIALS AND METHODS and ECM were found at 10–30 cm of soil depth. How- ever, prior to excising the root tips, the root systems were carefully washed to remove most of the adhering soil 2.1. The experimental plantation of P. pinea substrates. A total of 233 ECM (9 to 53 per plant) were taken (140 young ECM, 91 old ECM and two short roots The description of the experimental P. pinea planta- – which seemed non-mycorrhizal) using a binocular mi- tion in the Brasinvert’s domain (latitude: 43o 28’ 12"; croscope (table I). They were then washed once with o longitude: 4 18’ 52"; altitude: 1 m) was detailed by [2]. H2O2 during 30 seconds and then three times with o Two treatments were carried out: seedlings inoculated autoclaved H2O, and stored at –70 C for further DNA Dominance of R. rubescens in a P. pinea plantation 199 Table I. Number of plants and ECM examined and results of the successful PCR-ITS amplification and ITS-RFLP types obtained in the P. pinea plantation. Seasons Total of PCR of Y. ECM ITS-RFLP types & plants ECM [O. Y. ] T [s d t ] I II III IV V Spring C1/L2 281 [620]12[1200]00750 I2/L2 22 [19 3 ] 3 [1 2 0 ] 10002 Autumn I1/L1 20 [0 20] 17 [17 0 0 ] 11 0600 I2/L1 35 [13 22] 21 [8 12 1 ] 0 10 0 0 11 I3/L2 21 [12 9 ] 9 [0 9 0 ] 90000 I4/L2 9 [5 4 ] 0 [0 0 0 ] 00000 I5/L2 45 [23 22] 17 [3 14 0 ] 17 0000 I6/L2 53 [13 40] 39 [0 39 0 ] 39 0000 Total 233 [91 140] 118 [41 76 1 ] 77 10 13 5 13 ECM: ectomycorrhizae. O.: old. Y.: young. C and I: control and inoculated plants, respectively, with their corresponding numbers and lines (L) in the plot. T [s, d, t]: total of successful PCR amplifications [single, double, triple amplified DNA bands]. 1 Two short roots which seemed non-ectomycorrhizal were included. extraction and molecular analysis of the P. pinea EFC. Belgium) and 1.75 units Taq DNA polymerase The sampling approach described above presents three (GibcoBRL, Life Technologies) [12].